A major challenge in the development of novel cancer therapies has been a lack of genetic alterations whose altered protein products are easily druggable. Entire classes of cancer driver alterations such as transcription factor amplification or tumor suppressor gene mutation have failed to produce targeting therapeutics, despite decades of effort. Due to genomic instability, cancer cells frequently delete one copy of large segment of DNA containing essential genes. In fact, a median 10% of the genome is hemizygously deleted in cancer cells. We hypothesize that hemizygous loss of essential genes in cancer cells may cause selective vulnerabilities to their further suppression. These vulnerabilities are not shared by diploid cells composing normal tissues and may therefore form the basis of a therapeutic window. We have integrated genomic copy number data with the results of a genome-wide shRNA screen across 216 cancer cell lines to find 170 genes whose hemizygous deletion sensitizes cells to its further suppression with RNAi. One of the top hits in our analysis is the recently recognized oncogene, SF3B1 whose role in cancer remains unknown. SF3B1 is hemizygously deleted in approximately 21% of breast cancers. Our preliminary data support the hypothesis that hemizygous loss of SF3B1 (SF3B1loss) uniquely sensitizes cells to its further suppression. In Aim 1 we propose to further test the hypothesis that SF3B1 suppression selectively decreases the proliferation of SF3B1loss cancer cells using gene-editing technology and mouse models. We will test whether existing SF3b-targeting compounds can mimic the effects of RNAi to selectively kill SF3B1loss cells. In Aim 2, we will test the hypothesis that SF3B1 suppression causes apoptosis through splicing alterations in SF3B1loss cells. We will assess whether SF3B1loss cancer cells undergo alterations in cell cycle progression and apoptosis after SF3B1 suppression. We will use RNA-sequencing experiments to determine whether SF3B1loss cells undergo more dramatic alterations in pre-mRNA splicing upon knockdown of SF3B1 than cells with normal SF3B1 genomic copy number. In Aim 3, we will test the hypothesis that specific buffers account for the relative resistance o cells with normal SF3B1 genomic copy number to suppression of SF3B1. We will perform experiments to determine whether an excess of SF3B1 mRNA, protein or transcriptional reserve form the basis for the buffer that distinguishes cells with normal SF3B1 genomic copy number from cells with hemizygous loss in the face of SF3B1 suppression. The possibility that hemizygous deletion of essential genes represents a novel therapeutic window may enable the development of a large number of new cancer therapies. Our proposed study will form the basis for this effort and has the potential to extend targeted cancer therapy to many more cancer types and patients.